PROJECT SUMMARY/ABSTRACT This research project aims to investigate in vivo gain-of-function (GOF) mechanisms of the p53R245W hotspot mutation involved in initiation and progression of metastatic triple negative breast cancer. The genetic drivers of metastatic breast cancer, particularly triple negative breast cancer, are poorly understood. Approximately 54% of invasive breast cancers harbor a missense mutation in TP53, with the p53R248W hotspot mutation having the greatest association with poor survival. The Lozano lab has generated a somatic mouse model for p53R245W (murine counterpart to human p53R248W). The conditional allele expresses wild type p53, which converts to mutant p53 upon Cre recombinase activation. Ductal injection of adenovirus-Cre into the mouse mammary gland allows mutant p53 expression in a few cells surrounded by a normal stroma and immune system. Studies in our somatic breast cancer model indicates p53R245W mice give rise to a broad spectrum of breast tumors (n=25): Esr1-Pgr-Erbb2-(triple negative, 30.8%); Esr1-Pgr- Erbb2+(Her2-enriched, 38.4%) or Esr1/Pgr+Erbb2+ (luminal B, 30.8%) and 58% of these tumors become metastatic. The TNBC tumors generated in this model are of interest because 88% of human TNBCs harbor a p53 alteration. We hypothesize that p53R245W gain of function mechanisms cooperate with other genetic events leading to metastatic TNBC. We will use an integrated genomics approach to investigate how p53R245W contributes to breast cancer metastasis by (1) ChIP-seq and RNA-seq to discover transcriptional co-factor(s) and target genes mediating mutant p53 GOF and (2) isolation of circulating tumor cells followed by single cell copy number and RNA-seq analysis to explore cell survival and metastasis. There is a knowledge gap of the genomic landscape associated with mutant p53 and its role in the development of metastatic TNBC. Data from the proposed studies will provide a better understanding of the GOF mechanism of the p53R245W hotspot mutation and how it contributes to the evolution of metastatic TNBC. The proposed research is sponsored by Dr. Guillermina Lozano, an expert in p53 biology and genetically engineered mouse models, with extensive experience investigating the role of p53 and its pathway components in cancer. This research plan is co-sponsored by Dr. Nicholas Navin, an expert and early developer of single- cell sequencing, with experience implementing single-cell genomics to study the evolution of human breast cancers. The PI will have an Advisory Committee of additional experts including Dr. Michelle Barton and Dr. Jeffrey Rosen to provide mentoring during the award period. The primary objective of the training plan is to provide the PI with experience in cancer genetics, genomics, and mouse models to prepare her for a career as an independent investigator in breast cancer biology.